This invention relates to a process for the purification of peptides. In particular it relates to a chromatographic process for the preparative purification and formation of salts formed with inorganic acids of D-phenylalanyl-L-proline-L-arginal and structural variants thereof. The process comprises a preparative purification of the above noted peptides incorporating a salt exchange.
The tripeptide D-Phe-L-Pro-L-Arg-H and related tripeptides are unstable in the free base form owing to their tendency to undergo intramolecular cyclization. These peptides are disclosed by S. Bajusz et al. in U.S. Pat. Nos. 4,703,036 and 4,478,745, while the sulfate salt form of D-Phe-L-Pro-L-Arg-H is described by S. Bajusz et al. in U.S. Pat. No. 4,399,065. Copending application Ser. No. 07/589,553 filed Sep. 28, 1990 describes D-Phg-L-Pro-L-Arg-H and related compounds. These compounds have excellent activity in inhibiting the amidase activity of thrombin, and thus are useful in the prevention and control of the development of blood clots. The preparation of stable salt forms which are pharmaceutically acceptable is an important factor in the therapeutic use of these peptides.
During the preparation of the tripeptide numerous impurities can develop. For example, the formation of D-Phe-L-Pro-D-Arg-H as the incorrect arginine aldehyde isomer can occur. Also the D-Phe or D-Phg group can epimerize to form incorrect isomer and carboxy and hydroxymethyl side products are formed from the arginine portion during preparation. The levels of these undesirable side products are reduced during the process of this invention to acceptable levels.
Reversed Phase High Performance Liquid Chromatography, "RP-HPLC", is widely used in the purification of compounds. Typically, one uses a gradient of an aqueous phase and an organic phase for development of an HPLC chromatogram, for example, aqueous acetic acid or aqueous trifluoroacetic acid for the aqueous phase and an organic solvent or mixture of organic solvents such as acetonitrile, methanol, ethanol or THF. The stationary phase, or column support, may vary but generally an alkylsilane resin is used. In the process provided by this invention the aqueous phase of the gradient comprises an inorganic acid e.g. sulfuric acid or hydrochloric acid, and the peptide product is obtained as the salt formed with the acid. The acidic eluate of the chromatogram is treated with a water insoluble basic resin to neutralize the excess acid in the eluate. Separation of the resin from the eluate by filtration provides a solution of the purified peptide salt essentially free of inorganic salts. The water and acetonitrile are removed from the salt solution by freeze drying or by evaporation to provide the purified peptide salt in solid form.